Long-term expression is essential for virtually all gene therapy treatments of neurodegenerative disorders. Helper virus-free Herpes Simplex Virus (HSV-1) vectors, lentivirus vectors, or adeno- associated virus vectors can support recombinant gene expression for 6 to 14 months. Thus, significant improvements in long-term expression are required for human gene therapy. Our approach is to view the large, ~150 kb, genome of an HSV-1 vector as a minichromosome. We used boundary elements to create a euchromatin-like domain, and enhancers to turn on expression from specific promoters within this domain. During the previous grant period, we identified specific enhancers and promoters that support long- term expression. We isolated an upstream enhancer from the tyrosine hydroxylase promoter that supports long-term expression. We developed HSV-1 vectors that support inducible, long-term expression. We identified promoters that support glutamatergic or GABAergic neuron-specific, long- term expression. We developed targeted gene transfer to specific types of neurons, with long-term expression. We used these advances to support development of specific gene therapies. First, we corrected the rat model of Parkinson's disease by coexpressing three DA biosynthetic enzymes and a vesicular monoamine transporter (4-gene-vector). Expression in GABAergic striatal neurons was observed for 14 months. The 4-gene-vector supported higher levels of behavioral correction, higher levels of DA, and only this vector supported regulated release of DA. Second, we showed that genetic activation of protein kinase C (PKC) pathways in small groups of rat postrhinal cortex neurons enhances learning of visual object discriminations. Moreover, in aged rats, we showed that activation of PKC pathways in small groups of hippocampal dentate granule neurons corrects deficits in spatial learning. The goal of this proposal is to elucidate specific mechanisms that support long-term expression. The first specific aim will isolate and characterize specific enhancers, and their cognate enhancer binding proteins, that support long-term expression. The second specific aim will characterize the chromosomal state of HSV-1 vectors that support long-term expression. The third specific aim will overexpress specific enhancer binding proteins, or chromatin modifying enzymes, to improve long- term expression. Long-term recombinant gene expression is essential for virtually all gene therapy treatments of neurological diseases. During the previous grant period, we identified specific promoters that support long-term expression from Herpes Simplex Virus (HSV-1) vectors. This proposal will elucidate specific mechanisms that support long-term expression.